| Barley yellow dwarf viruses(BYDVs) are very important viral pathogens in cereal plants including barley, wheat and oat, solely transmitted to host plants by cereal aphids species in nature and causing yellow dwarf disease, which is considered to be one of the most important viral diseases worldwide because it significantly reduces the production and quality of cereals. In China, BYDVs mainly infect wheat and cause typically characteristic symptoms, such as foliar yellowing and reduction of tillering. One of the most important BYDVs in China is BYDV-GAV. For better control of yellow dwarf disease caused by BYDV-GAV in wheat, a better understanding of the mechanism of viral infection and virus-host interaction will facilitate the exploration of new approaches for viral disease control. However, the genome of wheat is very huge and the transformation system in wheat is still deficient, which makes the studies on molecular plant-virus interaction very difficult. Brachypodium distachyon is an emerging model plant for the functional genomics study of temperate grasses and cereals owing to its small size, simple genome, short life cycle and undemanding growth requirements. More importantly, B. distachyon possesses agronomic traits that are in common with wheat and shares high degrees of chromosomal synteny with wheat.Therefore, in this study, B. distachyon was chosen as a model host of BYDV-GAV to explore the mechanism of molecular interaction with its host plant. The results are as follows:(1) The model pathosystem of BYDV-GAV in B. distachyon was constructed. B. distachyon inbred line Bd21-3 plants were infected by BYDV-GAV via aphid inoculation. 21 days post inoculation, the inoculated plants showed conspicuous disease symptoms such as leaf reddening, dwarfness and root stunting, which are the typical disease symptoms of BYDVs in the most cereal crops. Virus accumulations in B. distachyon plants were confirmed by TAS-ELISA and RT-PCR. Thin sections were prepared from infected leaves and non-infected leaves for transmission electron microscopy observation. The results revealed that the viral particles were observed in the companion cells and that BYDV-GAV infection resulted in the deformation of chloroplasts of mesophyll cells. Additionally, an aphid-mediated transmission assay using B. distachyon plants as virus resource was also conducted, and the results indicated that BYDV-GAV could spread in B. distachyon plants. To compare the development of yellow dwarf disease between B. distachyon and susceptible wheat cultivar following BYDV-GAV infection during 21 days post inoculation, time course observation of symptoms was conducted and virus accumulation was analyzed using TAS-ELISA. The observation revealed that BYDV-GAV symptoms appear faster and more severe in B. distachyon than wheat plants, but the virus accumulation in B. distachyon is similar with wheat.(2) Host proteins interacted with BYDV-GAV were screened and characterized. Firstly, the cDNA entry library of B. distachyon was used to create a recombination-derived yeast two-hybrid(Y2H) library by LR reaction, and then the recombination-derived Y2 H library was amplified using semi-solid method. The final titer of cDNA library for Y2 H was 1.7×107 cfu/mL. Secondly, using Y2 H assay, the cDNA library of B. distachyon was screened with coat protein(CP), movement protein(MP) and RNAi suppressor(P6) of BYDV-GAV as baits. Bait plasmids were constructed using GatewayTM technology and the self-activation of three baits was tested. The results showed that none of the three baits had self-activation and 3-AT concentration of all baits for Y2 H was 50 mM. Thirdly, the cDNA library screening was performed with bait CP, MP and P6. Interactors first grew on the SC-Leu-Trp-His(50mM 3-AT) plates and then the expression of report genes(HIS3, URA3 and Lac Z) in these interactors were tested to determine the interactions. The prey plasmids in the determined interactors were isolated and sequenced. The results revealed that 16 proteins had weak interaction with CP, including 2 uncharacterized proteins, 5 chloroplast proteins(ATP-dependent 6-phosphofructokinase 5, transketolase, protein TIC110, chlorophyll a-b binding protein of LHCII type 1-like and ribulose bisphosphate carboxylase/oxygenase activase A), dynamin 2A-like, calmodulin-3, transmembrane protein 147 and so on. MP had 35 strong interactors screened from the library, including 4 proteins(14-3-3 like, transcription factor VOZ1, glycine-rich RNA-binding protein and 26 S proteasome non-ATPase regulatory subunit 1 homolog A-like protein) after sequence analysis. However, we have not obtained any proteins interacted with P6. At last, in order to verify the interactions, prey plasmids isolated from the interactors were re-transformed to the yeast cells harboring the corresponding bait plasmids. The results showed that transcription factor VOZ1 still had a strong interaction with MP.(3) Interaction between MP and VOZ1 was verified in vitro and in vivo. Firstly, GST pull-down assay was conducted to verify the interaction in vitro, using GST-MP and MBP-VOZ1 fusion expressed in Escherichia coli. Secondly, the subcellular localizations of MP and VOZ1 in Nicotiana benthamiana were determined by Agrobacterium-mediated inoculation. The results indicated that MP was localized in the nuclear envelope and the cytoplasm, while VOZ1 clustered in the cytoplasm. Lastly, using bimolecular fluorescence complementation(BiFC) assay combining with Agrobacterium-mediated inoculation, the interaction between MP and VOZ1 was verified in vivo. The localization of interaction was also determined after DAPI staining, indicating that MP interacted with VOZ1 in cytoplasm rather than nucleus.In conclusion, a pathosystem of BYDV-GAV in B. distachyon was developed. On the basis of the pathosystem, the proteins in B. distachyon that interacted with BYDV-GAV were screened and characterized in this study, which will facilitate the studies on the pathogenic mechanism of BYDV-GAV. |
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